Activated omentum becomes rich in factors that promote healing and tissue regeneration

In order to study the mechanism by which an omental pedicle promotes healing when applied to an injured site, we injected a foreign body into the abdominal cavity to activate the omentum. One week after the injection, we isolated the omentum and measured blood vessel density, blood content, growth and angiogenesis factors (VEGF and others), chemotactic factors (SDF-1α), and progenitor cells (CXCR-4, WT-1). We found that the native omentum, which consisted mostly of adipose tissue, expanded the mass of its non-adipose part (milky spots) 15– to 20-fold. VEGF and other growth factors increased by two– to four-fold, blood vessel density by three-fold, and blood content by two-fold. The activated omentum also showed increases in SDF-1α, CXCR-4, and WT-1 cells (factors and cells positively associated with tissue regeneration). Thus, we propose that an omentum activated by a foreign body (or by injury) greatly expands its milky-spot tissue and becomes rich in growth factors and progenitor cells that facilitate the healing and regeneration of injured tissue. This work was partly supported by a grant (no. 2000–241 to A.K.S.) from the Juvenile Diabetes Foundation International.     

Cellular basis of tissue regeneration by omentum

The omentum is a sheet-like tissue attached to the greater curvature of the stomach and contains secondary lymphoid organs called milky spots. The omentum has been used for its healing potential for over 100 years by transposing the omental pedicle to injured organs (omental transposition), but the mechanism by which omentum helps the healing process of damaged tissues is not well understood. Omental transposition promotes expansion of pancreatic islets, hepatocytes, embryonic kidney, and neurons. Omental cells (OCs) can be activated by foreign bodies in vivo. Once activated, they become a rich source for growth factors and express pluripotent stem cell markers. Moreover, OCs become engrafted in injured tissues suggesting that they might function as stem cells.Omentum consists of a variety of phenotypically and functionally distinctive cells. To understand the mechanism of tissue repair support by the omentum in more detail, we analyzed the cell subsets derived from the omentum on immune and inflammatory responses. Our data demonstrate that the omentum contains at least two groups of cells that support tissue repair, immunomodulatory myeloid derived suppressor cells and omnipotent stem cells that are indistinguishable from mesenchymal stem cells. Based on these data, we propose that the omentum is a designated organ for tissue repair and healing in response to foreign invasion and tissue damage.     

Isolation and in vitro characterization of pancreatic progenitor cells from the islets of diabetic monkey models

Recent studies on the identification of stem/progenitor cells within adult mouse and human pancreatic islets have raised the possibility that autologous transplantation might be used in treating type 1 diabetes. However, it is not yet known whether such stem/progenitor cells are impaired in type 1 diabetic patients or diabetic animal models. The latter would also allow us to test the efficacy of autologous transplantation in large animal models prior to clinical applications. The present study aims to determine the existence of stem/progenitor cells in the islets of diabetic monkey models and to assess the proliferation and differentiation potential of such cells in vitro. Our results indicate that there are pancreatic progenitor cells in the adult pancreatic islets in both normal and type 1 diabetic monkeys. The isolated pancreatic progenitor cells can be greatly expanded in culture. Upon the removal of growth medium, these cells spontaneously form islet-like cell clusters, which could be further induced to secrete insulin by inductive factors. Furthermore, the secretion of insulin and C-peptide from the islet-like cell clusters responds to glucose and other stimuli, indicating that the differentiated cells not only resemble beta-cells but also possess the unique biological function of beta-cells. This study provides a foundation for further characterization of adult pancreatic progenitor cells and autologous transplantation using pancreatic progenitor cells in treating diabetic monkeys.     

Transcranial electrical stimulation activates the reparative regeneration and insulin-producing function of beta-cells in alloxan-induced diabetic rats

In alloxan-induced diabetic rats, it was demonstrated that transcranial electrical stimulation of the brain endorphinergic structures activated the reparative regeneration of the damaged beta-cells of the Langerhans pancreatic islets. This was estimated on the histological sections of pancreas with hematoxylin-eosin staining. Several small newborn islets were found to originate from pancreatic progenitor cells. After transcranial electrical stimulation of insulin granules, beta-cells (Gomori's staining) were observed as an indication of the restoration of the insulin production. Correspondingly the increase of the blood insulin level was estimated by immune-enzyme method. The dynamics of the plasma insulin increase had a significant negative correlation with decrease of the blood glucose level. The glucose-lowering action of the transcranial electrical stimulation in alloxan-induced diabetic rats seems to be based on stimulation of the regeneration of damaged beta-cells with the restoration of their insulin production.     

Human iPS Cell-Derived Insulin Producing Cells Form Vascularized Organoids under the Kidney Capsules of Diabetic Mice

Type 1 diabetes (T1D) is caused by autoimmune disease that leads to the destruction of pancreatic β-cells. Transplantation of cadaveric pancreatic organs or pancreatic islets can restore normal physiology. However, there is a chronic shortage of cadaveric organs, limiting the treatment of the majority of patients on the pancreas transplantation waiting list. Here, we hypothesized that human iPS cells can be directly differentiated into insulin producing cells (IPCs) capable of secreting insulin. Using a series of pancreatic growth factors, we successfully generated iPS cells derived IPCs. Furthermore, to investigate the capability of these cells to secrete insulin in vivo, the differentiated cells were transplanted under the kidney capsules of diabetic immunodeficient mice. Serum glucose levels gradually declined to either normal or near normal levels over 150 days, suggesting that the IPCs were secreting insulin. In addition, using MRI, a 3D organoid appeared as a white patch on the transplanted kidneys but not on the control kidneys. These organoids showed neo-vascularization and stained positive for insulin and glucagon. All together, these data show that a pancreatic organ can be created in vivo providing evidence that iPS cells might be a novel option for the treatment of T1D.     

Islet cell engraftment and control of diabetes in rats after transplantation of pig pancreatic anlagen

The insufficient supply of tissue, loss posttransplantation, and limited potential for expansion of beta-cells restrict the use of islet allotransplantation for diabetes. A way to overcome the supply and expansion problems is to xenotransplant embryonic tissue. We have shown that whole rat pancreatic anlagen isotransplanted into the omentum of rats, or xenotransplanted into costimulatory blocked mice, undergo growth and differentiate into islets surrounded by stoma without exocrine tissue. Isotransplants normalize glucose tolerance in diabetic hosts. Here, we show that embryonic day 29 porcine pancreas transplanted into the omentum of adult diabetic rats undergoes endocrine tissue differentiation over 20 wk and normalizes body weights and glucose tolerance. Unlike rat-to-rodent transplants, individual alpha- and beta-cells engraft without a stromal component, and no immunosuppression is required for pig-to-rat transplants. Herein is described a novel means to effect the xenotransplantation of individual islet cells across a highly disparate barrier.     

Retinoic acid promotes the generation of pancreatic endocrine progenitor cells and their further differentiation into beta-cells

The identification of secreted factors that can selectively stimulate the generation of insulin producing beta-cells from stem and/or progenitor cells represent a significant step in the development of stem cell-based beta-cell replacement therapy. By elucidating the molecular mechanisms that regulate the generation of beta-cells during normal pancreatic development such putative factors may be identified. In the mouse, beta-cells increase markedly in numbers from embryonic day (e) 14.5 and onwards, but the extra-cellular signal(s) that promotes the selective generation of beta-cells at these stages remains to be identified. Here we show that the retinoic acid (RA) synthesizing enzyme Raldh1 is expressed in developing mouse and human pancreas at stages when beta-cells are generated. We also provide evidence that RA induces the generation of Ngn3(+) endocrine progenitor cells and stimulates their further differentiation into beta-cells by activating a program of cell differentiation that recapitulates the normal temporal program of beta-cell differentiation.     

Distribution of vasoactive intestinal polypeptide, neuropeptide-Y and substance P and their effects on insulin secretion from the in vitro pancreas of normal and diabetic rats

This study examined the pattern of distribution of vasoactive intestinal polypeptide (VIP), neuropeptide-Y (NPY) and substance P (SP) in the pancreas of diabetic rat to determine whether there are changes in the number and pattern of distribution of these neuropeptides after the onset of diabetes. Moreover, the effect of VIP, NPY and SP on insulin secretion from the pancreas of normal and diabetic rats was also examined. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (i.p.) (60 mg kg body weight(-1)). Four weeks after the induction of DM, diabetic (n = 6) and normal (n = 6) rats were anesthetized with chloral hydrate and their pancreases removed and processed for immunohistochemistry and insulin secretion. The number of insulin-positive cells in the islets of Langerhans was reduced while that of VIP and NPY increased significantly after the onset of diabetes. The pattern of distribution of VIP, NPY and SP in the nerves innervating the pancreas was similar in both normal and diabetic rats. VIP-evoked large and significant (P < 0.02) increases in insulin secretion from the pancreas of normal and diabetic rats. NPY also induced a marked (P < 0.005) increase in insulin release from pancreatic tissue fragments of normal rat. Stimulation of pancreatic tissue fragments of diabetic rat with NPY resulted in a slight but not significant increase in insulin release. SP induced a large and significant (P < 0.005) increase in insulin secretion from the pancreas of normal rat but inhibited insulin secretion significantly (P < 0.03) from isolated pancreas of diabetic rat. In summary, VIP and NPY can stimulate insulin secretion from the pancreas after the onset of diabetes. The stimulatory effect of SP on insulin secretion is reversed to inhibitory in diabetic rats.     

Histogenesis of the endocrine pancreas in newborn rats after destruction by streptozotocin. An immunocytochemical study

Endocrine pancreatic tissue in newborn rats was studied 1 to 17 days after the destruction of B cells by an injection of streptozotocin. Regeneration of insulin cells was observed four days after streptozotocin injection, which was followed by recovery from the diabetic state and an increased pancreatic insulin content. Regeneration was characterised by new islets budding from small ducts. The pancreas of newborn rats, like the embryonic pancreas, thus retains a capacity to form endocrine tissue, although some degree of reduplication of preexisting B cells may also be involved in the process. Newborn rats injected with streptozotocin constitute an interesting model for the study of factors which may act on the regenerative potential of pancreatic endocrine tissue in the diabetic state.     

Distribution of neurotransmitters and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues

The distribution of adrenergic, cholinergic and amino acid neurotransmitters and/or their enzymes were examined in both the normal and diabetic pancreatic tissues in rat using immunohistochemistry to determine whether changes in the pattern of distribution of nerves containing these neurotransmitters will occur as a result of diabetes mellitus. In addition to this, the effect of noradrenaline (NA), adrenaline (ADR), acetylcholine (ACh) and gamma-amino butyric acid (GABA) on glucagon secretion from the isolated normal and diabetic pancreatic tissues was also investigated. Pancreatic fragments from the tail end of normal and diabetic rats were removed and incubated with different concentrations (10(-8)-10(-4) M) of these neurotransmitters. Glucagon secretion into the supernatant was later determined by radioimmunoassay. NA at 10(-6) M evoked a three-fold increase in glucagon secretion from normal pancreatic tissue fragments. In diabetic pancreatic tissue, NA at 10(-6) M was able to increase glucagon secretion 1.5 times the value obtained from diabetic basal. ADR (10(-8) M) increased glucagon secretion slightly but not significantly in normal pancreatic tissue. ADR inhibited glucagon secretion from diabetic pancreas at all concentrations. ACh (10(-8) M) induced a five-fold increase in glucagon secretion from normal pancreatic tissue. In a similar way, ACh evoked a two-fold increase in glucagon secretion from diabetic pancreas at 10(-4) M. In normal pancreatic tissue, GABA produced a slight but not significant increase in glucagon secretion at 10(-4) M. In contrast to this it inhibited glucagon secretion from diabetic pancreatic tissue fragments at all concentrations. In summary, tyrosine hydroxylase- and choline acetyltransferase-positive nerves are equally well distributed in both normal and diabetic rat pancreas. There was an increase in the number of glucagon positive cells and a decrease in the number of GABA-positive cells in diabetic pancreas. NA and ACh have a potent stimulatory effect on glucagon secretion from normal pancreatic tissue fragments, whereas ADR and GABA produced a small but not significant increase in glucagon secretion from normal pancreas. NA and GABA stimulated glucagon secretion from diabetic pancreas. In contrast, ADR and ACh inhibited glucagon secretion from diabetic pancreas. Neurotransmitters vary in their ability to provoke glucagon secretion from either normal or diabetic pancreas.     

The role of leucine-enkephalin on insulin and glucagon secretion from pancreatic tissue fragments of normal and diabetic rats

Leucine-enkephalin (Leu-Enk) has been shown to be present in endocrine cells of the rat pancreas and may play a role in the modulation of hormone secretion from the islets of Langerhans. Since little is known about the effect of Leu-Enk on insulin and glucagon secretion, it was the aim of this study to determine the role of Leu-Enk on insulin and glucagon secretion from the isolated pancreatic tissue fragments of normal and diabetic rats. Pancreatic tissue fragments of normal and streptozotocin-induced diabetic rats were incubated for 1 h with different concentrations of Leu-Enk (10(-12)-10(-6)M) alone or in combination with either atropine or yohimbine or naloxone. After the incubation period the supernatant was assayed for insulin and glucagon using radioimmunoassay techniques. Leu-Enk (10(-12 )-10(-6)M) evoked large and significant increases in insulin secretion from the pancreas of normal rats. This Leu-Enk-evoked insulin release was significantly (p < 0.05) blocked by atropine, naloxone and yohimbine (all at 10(-6)M). In the same way, Leu-Enk at concentrations of 10(-12)M and 10(-9)M induced significant (p < 0.05) increases in glucagon release from the pancreas of normal rats. Atropine, yohimbine but not naloxone significantly (p < 0.05) inhibited Leu-Enk-evoked glucagon release from normal rat pancreas. In contrast, Leu-Enk failed to significantly stimulate insulin and glucagon secretion from the pancreas of diabetic rats. In conclusion, Leu-Enk stimulates insulin and glucagon secretion from the pancreas of normal rat through the cholinergic, alpha-2 adrenergic and opioid receptor pathways.     

Can pancreatic duct-derived progenitors be a source of islet regeneration?

The regenerative process of the pancreas is of interest because the main pathogenesis of diabetes mellitus is an inadequate number of insulin-producing β-cells. The functional mass of β-cells is decreased in type 1 diabetes, so replacing missing β-cells or triggering their regeneration may allow for improved type 1 diabetes treatment. Therefore, expansion of the β-cell mass from endogenous sources, either in vivo or in vitro, represents an area of increasing interest. The mechanism of islet regeneration remains poorly understood, but the identification of islet progenitor sources is critical for understanding β-cell regeneration. One potential source is the islet proper, via the dedifferentiation, proliferation, and redifferentiation of facultative progenitors residing within the islet. Neogenesis, or that the new pancreatic islets can derive from progenitor cells present within the ducts has been reported, but the existence and identity of the progenitor cells have been debated.In this review, we focus on pancreatic ductal cells, which are islet progenitors capable of differentiating into islet β-cells. Islet neogenesis, seen as budding of hormone-positive cells from the ductal epithelium, is considered to be one mechanism for normal islet growth after birth and in regeneration, and has suggested the presence of pancreatic stem cells. Numerous results support the neogenesis hypothesis, the evidence for the hypothesis in the adult comes primarily from morphological studies that have in common the production of damage to all or part of the pancreas, with consequent inflammation and repair. Although numerous studies support a ductal origin for new islets after birth, lineage-tracing experiments are considered the “gold standard” of proof. Lineage-tracing experiments show that pancreatic duct cells act as progenitors, giving rise to new islets after birth and after injury. The identification of differentiated pancreatic ductal cells as an in vivo progenitor for pancreatic β-cells has implications for a potentially important, expandable source of new islets for diabetic replenishment therapy.